These experiments have been initiated to characterize embryonic stem cells, especially human embryonic stem cells (hESC), especially for studies of human dopaminergic neuronal function. In spite of the great importance of dopaminergic neurons for drug abuse, in addition to their therapeutic potential via transplantation, there have been enormous obstacles to the direct study of human dopaminergic neurons. Until very recently such neurons have been obtainable only from human fetal material. The advent of human ES cells has made the derivation human dopaminergic neurons in vitro in unlimited quantities for research use a possibility. Our initial experiments were focused on mouse ES cells, and promoter systems which could be employed for transgene expression. It was found that the CMV and especially the EF1 promoter led to long-term stable transgene expression in ES cells. Interestingly, expression via both the EF1 and CMV promoter disappeared as the ES cells differentiated into dopaminergic neurons, using an established system for dopaminergic differentiation by stromal cell-mediated differentiation. In addition, promoters derived from the tyrosine hydroxylase and GAD65 genes are now being investigated. Since the aim of these studies involved primarily the use of human ES cells, and there was very little data in the literature concerning characterization of most of the lines in the NIH stem cell registry, we therefore determined to characterize the general properties of the hESC lines from BresaGen Inc., in terms of pluripotency, differentiation, stable maintenance, and gene expression patterns. Small and large scale oligonucleotide and cDNA arrays were also employed to characterize gene expression patterns in undifferentiated hESC. We have continued to employ mainly the BresaGen lines for studies of human embryonic stem cell differentiation. Experiments were also undertaken to obtain dopaminergic neuronal differentiation from hESC. It was found that co-culture with the stromal PA6 cell line induced dopaminergic differentiation with a defined and reproducible time course. Cells positive for tyrosine hydroxylase were first detected after 10 days of co-culture, with maximal numbers of cells positive for tyrosine hydroxylase being present after 20-23 days of co-culture. Differentiated dopaminergic neurons expressed a number of markers for mature dopaminergic neurons, including transcripts characteristic of neurotransmitter function and response to growth factors. Most colonies in each culture were found to contain dopaminergic cells after differentiation, although a minority of cells within each colony were dopaminergic and other cell types including non-CNS cells (e.g., cells positive for smooth muscle actin) were also present. After transplantation into the brain, some dopaminergic neurons were found to survive, although numbers of non-neuronal cells (e.g., smooth muscle actin positive cells) were much larger than numbers of CNS cells. Therefore, significant improvements will be needed before dopaminergic neurons derived from hESC can be employed for therapeutic transplantation. In addition, a number of experiments were performed to identify the factors which are responsible for stromal cell-mediated differentiation. Factors identified as possible contributors include hepatocyte growth factor and FGF8;however, these factors alone are not sufficient to induce dopaminergic differentiation. We have currently initiated a larger effort to identify the specific factors which can induce dopaminergic differentiation from hESC. We have also identified a variant hESC line, BG01V, with karyotypic abnormalities, which can be grown more easily than the normal BG01 hESC line, but which undergoes dopaminergic differentiation with the same pattern and time course as BG01. This cell line may be very useful for identifying the factors responsible for stromal cell-mediated dopaminergic differentiation. Experiments were conducted using microarrays to identify the protein products of stromal cells which are responsible for dopaminergic differentiation of hESC. Using the karyotypically abnormal variant of BG01 cells, as well as karyotypically-normal hESC, we have identified a novel group of four factors which, when applied to hESC in combination, leads to differentiation of dopaminergic neurons with a midbrain identity directly from hESC. The resultant neurons exhibit action potentials, form synaptic connections, and express a number of proteins which are associated with mesencephalic dopamine-producing neurons. The four factors, collectively termed "SPIE", are stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). Omission of any one factor results in a loss of activity. We have recently also completed a comprehensive characterization of gene expression in neural stem cells and dopaminergic neurons during differentiation from hESC using the Massive Parallel Signature Sequencing (MPSS) technique. For these experiments, differentiating dopaminergic neurons were isolated by fluorescent-activated cell sorting on the basis of polysialyated N-CAM expression on cell surfaces. These sorted cells were found to be capable of complete differentiation, as indicated by neuronal function defined by electrophysiological criteria. A number of novel transcriptional changes associated with dopaminergic differentiation were identified, including a pronounced activation of a cluster of genes located at chromosome 11p15.5, a region which also includes the insulin and tyrosine hydroxylase genes. Current experiments are also focused on developing genetically altered cell lines for specific purposes, such as monitoring the status of differentiation via reporter transgenes, and employing hESC-derived dopaminergic neurons to characterize the effects of drugs of abuse on human dopaminergic neuronal function.